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研究生:黃于玲
研究生(外文):Yu-Ling Huang
論文名稱:利用高通量定序技術分析神經突觸內mRNA之分布
論文名稱(外文):Transcriptome analysis of mRNAs localized to synapses by high-throughput sequencing
指導教授:戴桓青戴桓青引用關係
指導教授(外文):Hwan-Ching Tai
口試委員:黃憲松陳平
口試委員(外文):Hsien-Sung HaungRichard Ping Cheng
口試日期:2018-07-13
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2018
畢業學年度:107
語文別:英文
論文頁數:69
中文關鍵詞:阿茲海默症突觸體mRNA定位次世代定序流式細胞儀蔗糖梯度
DOI:10.6342/NTU201802050
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許多神經系統疾病例如癡呆症,自閉症,精神分裂症,精神發育遲滯等疾病的根本原因皆與突觸功能障礙有關。其中,最常見的老年性癡呆症─阿茲海默症,其慢性的神經退化過程中會伴隨著神經突觸的喪失,進而導致神經凋亡。與老年斑、神經元纖維纏結、β-澱粉樣蛋白斑、過度磷酸化的Tau蛋白等相比,突觸喪失與阿茲海默症中的認知能力下降更有密切的關聯性。阿茲海默症的致病機制主要與三種重要的蛋白質相關,分別為β-澱粉樣蛋白、Tau蛋白和ApoE載脂蛋白,而這三種蛋白皆為神經突觸蛋白。
當人們將阿茲海默症的病徵研究著重在位於突觸末梢表現異常的蛋白質時,更值得注意的是在神經及突觸的末端亦存在著大量的RNA分子,包含了mRNA、microRNA、以及長鏈非編碼 RNA。這些突觸通常含有多核醣體,蛋白質轉譯也在突觸內發生,這些新合成的蛋白質在突觸的可塑性上扮演了相當重要的角色。然而,哺乳動物大腦中典型的興奮性突觸非常小,直徑只有約500奈米,且它們僅有非常低的mRNA拷貝數。因此,通過傳統的螢光原位雜交技術(FISH)鑑定定位於突觸的mRNAs是非常具有挑戰性的,再加上若要以FISH鑑定數千種基因在突觸中的功能定位,實際上是難以實行的。
為此我們開發了一種方法,利用次世代測序(NGS)的靈敏度和高通量以確定突觸末端的mRNA組成。我們通過簡單的沉澱法(粗製備方法)和蔗糖梯度離心法(蔗糖製備方法)來製備具有較高濃度突觸的樣品,從非常少量的小鼠皮質突觸末端作為初始材料,我們利用設計用於單細胞的高靈敏度轉錄組擴增試劑組將這些突觸的mRNA轉換為cDNA文庫。綜上所述,我們通過對小鼠基因組進行生物資訊比對,約可獲得500個轉錄體,再使用幾種生物信息學方法分析這些基因的關係,包括基因本體論和同源群簇等方法,發現這些基因主要與主要與突觸,細胞外囊泡,核糖核蛋白和線粒體有關。
我們從次世代測序中一共獲得約16億次讀數,約有9.5%的比對率。根據結果顯示,相較於粗製備法,蔗糖梯度製備法可獲得更多的突觸,雖然兩種方法所製備的樣品仍會含有其他胞器導致樣品有其他污染。此外,低比對率顯示出小鼠蛋白質數據庫仍不足以完整分析小鼠轉錄組,因為轉錄組中可能含有其他表現異常的的基因異構物和選擇性剪接上之變異。因此,在未來我們將利用螢光染色來進行分選以獲取高純度突觸末端,並通過建立客製化基因數據庫進行更深入的轉錄組分析。
Synaptic dysfunction underlies many neurological disorders such as dementia, autism, schizophrenia, mental retardation, etc. In Alzheimer’s disease (AD), the most common form of senile dementia, synaptic loss precedes neuronal death in the prolonged process of neurodegeneration. The loss of synapse shows a very strong correlation with cognitive decline in AD, stronger than the levels of senile plaques, neurofibrillary tangles, beta-amyloid (Aβ) oligomers, or hyperphosphorylated tau. There are three important proteins involved in the pathogenesis of AD—Aβ, tau, and apolipoprotein E, all of which are synaptic proteins.
While much attention has been paid to protein abnormalities at synaptic terminals, it should also be noted that nerve endings and synaptic terminals also contain numerous RNA molecules. These include mRNAs, microRNAs, and long non-coding RNAs. The synapse where proteins are translated often contains polyribosomes, and the newly synthesized proteins play critical roles in synaptic plasticity. However, the typical excitatory synapses in mammalian brains are very small, only about 500 nm in diameter, and they contain a very low copy number of mRNAs. It is therefore extremely challenging to identify mRNAs localized to synapses by conventional fluorescence in situ hybridization (FISH) techniques. To optimize FISH assays for thousands of genes to probe their synaptic localization is largely impractical.
We devised a strategy to identify the mRNA composition of synaptic terminals that takes advantage of the sensitivity and high throughput of next generation sequencing (NGS). We prepared synaptically enriched biochemical fractions by simple sedimentation (crude preparation) and by sucrose gradient centrifugation (sucrose preparation). Starting with very small amounts of mouse cortical synaptic terminals, we utilized highly sensitive transcriptome amplification kits designed for single cells to convert their mRNAs into cDNA libraries. Altogether, we identified about 500 transcripts in these synaptic preparations by blasting against mouse genome. Several bioinformatics methods were used to analyze the relationship of these genes, including Gene Ontology and Clusters of Orthologous Groups. They are mostly associated with synapses, extracellular vesicles, ribonucleoproteins, and mitochondria.
In total, we obtained about 1.6 billion reads from sequencing runs with 9.5% alignment rate after BLAST. The sucrose preparation shows better synaptic enrichment than the crude preparation but both still contain various contaminating organelles. The low alignment rate suggests that mouse protein databases are insufficient for the full analysis for the mouse transcriptome, which may express unusual gene isoforms and alternative splicing variants. In the future, we will acquire high-purity synaptic terminals by fluorescence activated cell sorting and carry out deeper transcriptome analysis by building custom gene databases.
誌謝 i
中文摘要 ii
ABSTRACT iv
CONTENTS vi
LIST OF FIGURES ix
LIST OF TABLES x
Abbreviations xi
Chapter 1 Introduction 1
1.1 An Overview of the Brain 1
1.2 The synapse 3
1.3 Synaptic dysfunction in Alzheimer''s disease 6
1.4 Localization of mRNA in synapse 11
1.5 Isolation of synaptosome 15
1.6 Flow cytometry of synaptosome 16
1.7 Procedures for RNA extraction and quality control 17
1.8 Next generation sequencing 19
1.9 Aim of study 21
Chapter 2 Materials and Methods 22
2.1 Materials 22
2.1.1 Mouse brains 22
2.1.2 Chemicals, consumables, materials and kits 22
2.1.3 Buffers 23
2.2 Instruments 24
2.2.1 BD FACS CantoII 24
2.2.2 CFX96 Touch Real-Time PCR Detection System 25
2.3 Subcellular fractionation of mouse brain 26
2.3.1 Crude synaptosome preparation 26
2.3.2 Synaptoneurosomes preparation with sucrose gradient 26
2.4 Flow cytometry for counting synapses 27
2.5 RNA extraction and double-strand cDNA synthesis 27
2.6 Quality control by RT-qPCR and Bioanalyzer 28
2.7 RNA sequencing and data analysis 28
Chapter 3 Results and Discussion 29
3.1 Qualification of extracted mRNA and cDNA libraries 29
3.2 The assembly status of RNA sequencing 33
3.3 The efficiency of two isolation methods 37
3.4 Brief function classification of identified transcriptomes 39
3.5 A deeper look at synaptic mRNA localization 42
3.5.1 Visualization of molecular interaction networks 42
3.5.2 The differential expression analysis 45
3.6 Comparison with prior studies 46
Chapter 4 Conclusions 49
4.1 Comparisons of two isolation methods 49
4.2 Database construction with identified synaptic mRNA 49
4.3 Future research on isolation method and customized sequencing 49
REFERENCE 50
APPENDIX 61
1.1 Preparing mouse crude synaptosome 61
1.2 Mouse synaptoneurosomes preparation 63
1.3 Sucrose gradient isolation 65
1.4 REPLI-g WTA single cell kit protocol 67
1.5 RT-qPCR protocol 69
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